Food Chemistry 135 (2012) 1592–1599

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Food Chemistry

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Prebiotic effects of yacon (Smallanthus sonchifolius Poepp. & Endl), a sourceof fructooligosaccharides and phenolic compounds with antioxidant activity

David Campos a,⇑, Indira Betalleluz-Pallardel a, Rosana Chirinos a, Ana Aguilar-Galvez a, Giuliana Noratto b,Romina Pedreschi c

a Instituto de Biotecnología (IBT), Universidad Nacional Agraria La Molina – UNALM, Av. La Molina s/n, Lima, Perub Department of Nutrition and Food Science, Texas A&M University, College Station, Texas 77843, United States Institute for Obesity Research and Program Evaluation, Texas A&MAgriLife Research, College Station, TX 77843, United Statesc Food & Biobased Research, Wageningen UR. Bornse Weilanden 9, 6708WG, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 February 2012Received in revised form 25 April 2012Accepted 23 May 2012Available online 30 May 2012

Keywords:YaconSmallanthus sonchifoliusAccessionsPhenolic compoundsAntioxidant activityFructooligosaccharides

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.05.088

⇑ Corresponding author. Tel./fax: +511 3495764.E-mail address: dcampos@lamolina.edu.pe (D. Cam

Thirty-five different yacon (Smallanthus sonchifolius Poepp. & Endl) accessions were evaluated as potentialalternative sources of fructooligosaccharides (FOS) and phenolic type natural antioxidants. FOS, totalphenolics (TPC) and antioxidant capacity (AC) contents in the ranges of 6.4–65 g/100 g of dry mater(DM), 7.9–30.8 mg chlorogenic acid (CAE)/g of DM and 23–136 lmol trolox equivalente (TE)/g DM werefound. Accession AJC 5189 sparked attention for its high FOS content while DPA 07011 for its high TPCand AC. In addition, the prebiotic effect of yacon FOS was tested in vivo with a guinea pig model. A dietrich in yacon FOS promoted the growth of bifidobacteria and lactobacilli, resulting in high levels of shortchain fatty acids (SCFAs) in the cecal material and enhancement of cell density and crypt formation incaecum tissue, being indicative of colon health benefits. This study allowed identification of yacon culti-vars rich in FOS, AC and/or FOS and AC for nutraceutical applications.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Yacon (Smallanthus sonchifolius Poepp. & Endl), an Andean cropthat grows at altitudes of 1000–3200 m above sea level, is particu-larly known as an abundant source of b-(2 ? 1) fructooligosaccha-rides (FOS) (Goto, Fukai, Hikida, Nanjo, & Hara, 1995). FOS areconsidered as prebiotics and yacon FOS prebiotic effects have beendemonstrated in vitro showing that they were selectively fer-mented by bifidobacteria and lactobacilli (Pedreschi, Campos, Nor-atto, Chirinos, & Cisneros-Zevallos, 2003). In addition, yacon rootsare rich in phenolic compounds, mainly chlorogenic (caffeoyl-qui-nic) acid and other caffeic acid derivatives (Takenaka et al., 2003;Yan et al., 1999).

Yacon roots have a long history of safe use in South America andelsewhere with potential health-promoting properties, includingprebiotic, antidiabetic, antioxidative and antimicrobial effects(Ojansivu, Ferreira, & Salminen, 2011). Yacon cultivation has beenexpanded to several countries such as New Zealand, Japan, and Brazilin the last decades, and the production in the Andean region andother countries have increased due to the presumed medicinal prop-erties of both roots and leaves (Genta, Cabrera, Grau, & Sánchez,

ll rights reserved.

pos).

2005). The antidiabetic effects of yacon root hydroalcoholic extractin streptozotocin (STZ)-induced diabetic rats have been attributedto its antioxidant activity and content in phenolic compounds,mainly chlorogenic acid (Park, Yang, Hwang, Yoo, & Han, 2009). Dailyintake of yacon syrup decreased body weight, waist circumferenceand body mass index suggesting a role in obesity management. Inaddition, beneficial effects have been reported on insulin resistanceand serum LDL-cholesterol levels suggesting a role on metabolicsyndrome and diabetes (Genta et al., 2009). Stimulatory effects of ya-con FOS on Ca intestinal absorption, bone mineral retention andstructural properties in the femoral midshaft of Wistar rats fed adlibitum with diets supplemented with yacon flour, have also beenreported (Lobo, Colli, Alvares, & Filisetti, 2007). Most of the beneficialeffects of yacon consumption have been attributed to its content ofphenolic compounds, antioxidants and prebiotics (FOS).

The largest diversity of yacon germoplasm is mainly found in theeastern Andean slopes of Peru and Bolivia (Grau & Rea, 1997). Up todate, only a few studies have related the biodiversity to the physicaland chemical characteristics of yacon. Large differences in FOS andsugar contents have been reported for ten different yacon accessions(Hermann, Freire, & Pazos, 1997). There are also differences in tubershape, weight, content of oligofructans, as well as in leaf isozymes,phenolics, and relative DNA contents reported for four yacon acces-sions cultivated under field conditions (Valentová et al., 2006). In an

D. Campos et al. / Food Chemistry 135 (2012) 1592–1599 1593

effort to investigate the intra-specific genetic variability in S.sonchifolius for prediction of its total phenolic content, molecularmarkers have recently been investigated (Milella et al., 2011).

The objectives of this study are: (i) to evaluate total phenoliccompounds (TPC), antioxidant capacity (AC), reducing sugars(RS), sucrose and FOS in 35 yacon accessions to identify accessionswith potential to be used as sources of prebiotics and other bioac-tive compounds and (ii) to investigate in vivo the prebiotic effectsof yacon FOS compared to the gold standard ‘inulin’.

Table 1Formulation of experimental diets (g/100 g).

Ingredients Diet control Diet inulina Diet yacon floura

Yellow maize 28.06 27.97 25.28Wheat by-product 34.95 34.79 30.84Rice husk 5.00 0.00 0.00Inulin (Orafti�P95) 0.00 5.26 0.00Yacon flour 0.00 0.00 11.9Alfalfa hey 6.1 6.1 6.1Soya cake 20.62 20.61 20.61Vegetal oil 2.35 2.35 2.35Mix (vitamins + minerals) 2.76 2.76 2.76Anti-fungal 0.1 0.1 0.1Vitamin C 0.06 0.06 0.06

Diet Composition: protein 18.0 (%), fibre 9.0 (%), fat 5.0 (%), lysine 0.84 (%),methionine + cysteine 0.79 (%), arginine 1.39 (%), tryptophan 0.28 (%), treonine 0.69(%), calcium 0.80 (%), phosphorus 0.80 (%), sodium 0.20 (%) vitamin C 200 (mg/100 g). Gross energy 12.14 (Mj/kg).

a The content of inulin or FOS in the diet was 5 %.

2. Materials and methods

2.1. Plant material and chemicals

Thirty-five accessions of yacon roots were kindly supplied bythe International Potato Center (CIP) located in Lima (Peru). Theroots were grown in the region of Huancayo (Peru) at approxi-mately 3200 m above sea level, at �80% relative humidity and17 �C (average temperature). Three independent samples of�0.5 kg were collected for each accession. Samples were collectedat optimal harvest date (8 months of cultivation) and stored at�20 �C for further use. Moisture content was determined in yaconflesh and DM was calculated by difference (AOAC, 1995).

The standards used for analysis of phenolic acids (p-coumaric,o-coumaric, protocatechuic, ferulic, gallic, caffeic, chlorogenic andp-hydroxybenzoic acids), flavonols (quercetin, rutin, myricetinand kaempherol), flavones (luteolin and apigenin) and flavanones(naringenin) were purchased from Sigma Chemical Co. (St. Louis,MO). Flavan-3-ols (cathechin and epicathechin) were purchasedfrom ChromaDexTM (Santa Clara, CA). Trolox (6-hydroxy-2,5,7,8-tetramethyl chroman- 2-carboxylic acid) and 2N Folin–Ciocalteureagent, 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) were purchased from Sigma Chemicals Co. (St. Louis, MO).HPLC grade acetonitrile and other solvents and reagents were pur-chased from Merck (Darmstadt, Germany).

2.2. Chemical analysis

2.2.1. Antioxidant capacity (AC) and total phenolic compounds (TPC)Yacon flesh (�5.0 g) was homogenized with 100 ml of acidified

80% methanol (formic acid, pH 2.0). The mixture was vortexed for30 s and flushed with nitrogen for 2 min. After 60 min of intermit-tent shaking (200 rpm) at room temperature, the extract was cen-trifuged at 6000g for 10 min at 4 �C, and the supernatant wascollected. The pellet was submitted to a second extraction for30 min with 50 ml of solvent. The supernatants were combinedand evaporated on a rotary evaporator at 40 �C for further AC,TPC, and HPLC-PAD (photodiode array detection) analysis.

AC was determined using the ABTS assay (Arnao, Cano, & Aco-sta, 2001) and expressed as lmol of trolox equivalents (TE)/g ofDM from a standard curve developed with trolox. TPC were deter-mined using a standard curve of chlorogenic acid (CA) according toSingleton and Rossi (1965) and the results were expressed as mil-ligram of chlorogenic acid equivalents (CAE)/g of DM.

2.2.2. HPLC-PAD analysis of phenolic compoundsPhenolic compound profiles were analyzed by HPLC-PAD as

previously reported (Chirinos et al., 2008). Briefly, phenolics wereseparated using a X-terra RP-C18 (5 lm, 250 � 4.6 mm) column(Waters, Milford, MA) and a 2.0 � 4.6 mm guard column at 30 �Con a Waters 2695 Separation Module (Waters, Milford, MA)equipped with an auto-injector, a 2996 photodiode array detector(PDA) and the Empower software. Spectral data were recordedfrom 200 to 700 nm. The mobile phase was solvent (A) water: ace-tic acid (94:6, v/v, pH 2.2) and solvent (B) acetonitrile. The solvent

gradient was: 0–15% B in 40 min, 15–45% B in 40 min and 45–100%B in 10 min. A flow rate of 0.5 ml/min was used and 20 ll of samplewere injected. Samples and mobile phases were filtered through a0.22 lm Millipore filter, type GV (Millipore, Bedford, MA) prior toHPLC injection. Each sample was analyzed in duplicate. Phenoliccompounds were identified and quantified by comparing theirretention time and UV–Vis spectral data to standards.

2.2.3. Quantitate of reducing sugars, FOS and sucroseSugars and FOS were extracted from yacon flesh according to the

method reported by Jaime, Martín-Cabrejas, Mollá, López-Andréu,& Esteban, 2001 with some modifications (Jaime et al., 2001).Briefly, 5 g of yacon was homogenized in an ultra-turrax homoge-nizer (IKA, Germany) with 50 ml of 70% ethanol (v/v) and immedi-ately heated at 100 �C for 10 min. The mixture was centrifuged at6000g for 15 min and the supernatant was collected. The yaconcakes were re-extracted two more times under the same conditions.

The supernatants were combined and vacuum evaporated in arotary evaporator at 50 �C. The residue was re-dissolved in 50 mlof deionized water, and this aqueous extract was kept for furtheranalysis of reducing sugars (RS), sucrose (S) and fructooligosaccha-rides (FOS). RS were determined according to the method reportedby Miller (1959) using dinitrosalicilic acid as reagent and fructoseas standard, absorbance was measured at 550 nm. FOS content wasdetermined by HPLC-IR as reported by Campos, Betalleluz, Tauqu-ino, Chirinos and Pedreschi (2009). Briefly, a NH2P-50 4E (5 lm,250 � 4.6 mm) column (Shodex, Japan) and a NH2P-50G 4A(4.6 � 10 mm) guard column were used. The mobile phase wascomposed of water: acetonitrile (30:70, v/v) at a flow rate of1 ml/min and 30 �C. Twenty ml of sample was injected. Samples,standards (glucose, fructose and sucrose) and mobile phase werefiltered through a 0.22 lm Millipore filter, type GV (Millipore,Bedford, MA) prior to HPLC injection. The results were expressedas g of fructans per 100 g of DM.

2.3. Prebiotic effects of yacon FOS using an in vivo guinea pig model

Yacon roots were obtained from a local market in Lima, Peru.Roots were boiled (99.5 �C for 25 min) to inactive enzymes, cut intoslices and dried (�7% moisture) using a hot-air tunnel (relativehumidity of�22% and air flow rate of 2.5 m/s) at 65 �C. Dried yaconslices were milled to obtain yacon flour composed of FOS, 42%; RS33% and sucrose 4.9% for further use in the diet formulation pre-sented in Table 1. Pellets were obtained using a Hessen Boxtel–Holland (Robinson Milling Systems, Lima, Peru) machine.

Male guinea pigs (Cavia porcellus) of 14 ± 2 days age and230 ± 30 g weight were purchased from a commercial guinea pig

Table 2Total phenolic compounds, antioxidant capacity, dry matter, reducing sugars, sucrose and fructooligosaccharides in 35 yacon accessions.

Cultivar TPC (mg CAE/g DM) AC (lmol TE/g DM) D M (g/100 g) R S (g/100 g DM) S (g/100 g DM) FOS (g/100 g DM)

ARV 5073 9.0 ± 0.3 43.3 ± 2.4 15.1 ± 1.3 32.8 ± 0.8 7.1 ± 0.2 29.7 ± 2.3ARB 5564 8.9 ± 1.2 42.5 ± 5.0 11.0 ± 0.2 32.3 ± 0.1 5.1 ± 0.9 41.5 ± 3.6ARB 5185 10.3 ± 1.0 43.5 ± 6.5 14.2 ± 1.4 36.8 ± 3.2 4.8 ± 0.1 32.4 ± 4.7AJC 5189 7.9 ± 0.8 45.4 ± 6.0 19.1 ± 1.2 19.7 ± 0.3 2.6 ± 0.2 65.0 ± 2.0ARB 5184 12.0 ± 0.4 40.4 ± 4.7 9.1 ± 0.4 39.0 ± 2.8 6.4 ± 0.4 30.2 ± 2.8DPA 07008 11.7 ± 0.5 39.5 ± 1.3 11.5 ± 0.8 43.1 ± 6.1 4.4 ± 0.2 47.2 ± 4.3AMM 5163 10.9 ± 0.7 52.6 ± 4.4 18.6 ± 1.2 26.3 ± 2.6 4.0 ± 0.0 49.7 ± 7.5ARB 5027 18.2 ± 1.2 69.8 ± 0.1 10.3 ± 0.9 43.3 ± 1.1 7.2 ± 0.8 37.5 ± 2.5DPA 07010 11.8 ± 1.3 88.4 ± 1.2 7.5 ± 0.2 49.1 ± 3.9 8.9 ± 0.4 38.5 ± 0.2DPA 07004 14.9 ± 0.8 57.3 ± 1.8 10.2 ± 0.1 43.1 ± 2.5 4.8 ± 0.6 28.4 ± 0.3ARB 5382 13.0 ± 0.9 38.7 ± 5.1 11.3 ± 0.7 52.7 ± 2.2 2.2 ± 0.6 21.4 ± 2.8DPA 07007 10.0 ± 0.8 59.6 ± 0.1 12.5 ± 1.4 45.8 ± 1.4 3.7 ± 0.1 38.6 ± 1.1ARB 5125 10.3 ± 0.4 23.3 ± 2.5 13.6 ± 0.2 27.3 ± 3.2 2.0 ± 0.1 47.1 ± 4.5AKW 5075 11.0 ± 0.4 38.1 ± 2.3 17.1 ± 0.7 30.8 ± 0.8 3.3 ± 0.7 51.3 ± 3.6ARB 5124 11.3 ± 1.3 60.4 ± 3.4 17.0 ± 0.6 28.4 ± 3.4 5.9 ± 1.6 47.0 ± 2.0DPA 7001 26.3 ± 0.3 122.0 ± 0.1 10.6 ± 0.0 56.1 ± 3.1 16.8 ± 0.5 12.2 ± 0.5DPA 7005 25.8 ± 0.1 133.8 ± 2.9 12.2 ± 0.2 59.9 ± 1.7 6.1 ± 0.5 20.0 ± 0.2DPA 7006 24.0 ± 0.1 110.7 ± 1.7 13.0 ± 0.5 55.4 ± 1.0 5.9 ± 0.1 22.6 ± 0.0DPA 7002 24.5 ± 0.0 112.3 ± 0.1 12.7 ± 0.3 45.7 ± 0.4 14.1 ± 0.3 17.0 ± 0.6ARB 5563 22.3 ± 0.1 136.0 ± 6.1 14.8 ± 0.0 42.7 ± 0.5 7.2 ± 1.4 34.4 ± 0.0P 1385 14.3 ± 0.4 72.5 ± 5.4 16.2 ± 0.1 45.6 ± 0.7 2.0 ± 0.1 32.9 ± 0.5AMM 5129 12.4 ± 0.2 57.9 ± 6.4 15.7 ± 0.6 33.2 ± 0.4 2.1 ± 0.0 45.1 ± 1.3DPA 7009 26.0 ± 0.0 118.4 ± 0.2 12.0 ± 1.3 59.1 ± 0.4 4.2 ± 0.3 29.3 ± 1.3SAL 136 14.9 ± 0.5 73.3 ± 2.8 15.3 ± 0.2 45.2 ± 1.0 5.8 ± 0.2 38.2 ± 4.3Y. MORA. 11.2 ± 0.3 67.8 ± 0.1 13.1 ± 0.0 48.2 ± 0.0 6.9 ± 0.4 27.9 ± 2.1ARB 5537 22.6 ± 0.2 83.2 ± 2.6 13.6 ± 0.1 54.9 ± 0.7 7.9 ± 1.2 26.4 ± 1.5P 1185 20.6 ± 0.0 83.8 ± 6.1 13.0 ± 0.0 58.7 ± 0.5 5.0 ± 0.3 20.4 ± 0.9GOM 130 19.7 ± 0.2 91.8 ± 4.6 14.6 ± 0.0 48.7 ± 0.7 7.3 ± 0.1 32.8 ± 0.2AMM 5135 19.7 ± 0.2 99.2 ± 7.2 16.3 ± 0.1 45.3 ± 0.0 4.9 ± 0.1 42.0 ± 0.6Y. BLANCO 28.3 ± 0.1 135.7 ± 4.5 11.1 ± 0.1 67.5 ± 0.0 5.2 ± 0.0 19.3 ± 0.6AME 5186 25.6 ± 0.0 95.9 ± 7.2 11.0 ± 0.0 75.9 ± 4.3 6.6 ± 1.5 6.4 ± 2.5AMM 5150 18.9 ± 0.1 92.2 ± 6.1 15.5 ± 0.0 51.6 ± 0.4 6.5 ± 0.5 31.3 ± 0.3AMM 5136 22.4 ± 0.0 111.6 ± 4.3 13.6 ± 0.0 75.1 ± 1.6 8.3 ± 0.2 23.8 ± 0.1DPA 07011 30.8 ± 0.1 135.1 ± 0.1 10.1 ± 0.0 66.0 ± 3.5 8.0 ± 1.2 6.9 ± 0.7DPA 7003 23.3 ± 0.1 124.0 ± 4.4 13.3 ± 0.0 56.1 ± 0.1 4.8 ± 0.0 25.8 ± 0.1

Values are mean (n = 3) ± SD.

Fig. 1. HPLC-PAD phenolic compounds profile for yacon root accession DPA 07011 at 320 nm.

1594 D. Campos et al. / Food Chemistry 135 (2012) 1592–1599

farm (Universidad Nacional Agraria La Molina, Cieneguilla, Perú).The guinea pigs were kept in ventilated cages covered with ricepeel. Food and water were provided ad libitum. Animals were al-lowed to adjust to their environment for 4 days before initiationof the experiments and randomly assigned to three experimentalgroups (n = 16). Experimental groups received a basal diet (controlgroup), a diet with inulin Orafti� P95 (positive control group) or adiet with yacon flour (Table 1). Food intake and body weight wererecorded weekly during eight weeks. Body weight gain and feedefficiency (ratio of weight gain to food consumption) were calcu-

lated. At the end of the study (8 weeks), animals were humanelysacrificed using ether anesthesia following institutional regula-tions. Caecum was removed and the cecal content collected andfrozen in liquid nitrogen. Cecal material and caecum tissues werestored at �80 �C for further analysis.

2.3.1. Bacteriological analysis of cecal materialOne gram of cecal material was transferred into a sterile tube and

mixed with 9 ml of sterile saline phosphate solution (PBS, Sigma Al-drich) containing 1% of hydrochloride L-cysteine (Scharlau Chemie,

D. Campos et al. / Food Chemistry 135 (2012) 1592–1599 1595

Barcelona, Spain) and then serially diluted (from 10�1 to 10�7). Bif-idobacteria were quantified using Beerens medium (brain heartinfusion agar Difco™, glucose, citrate of iron III, L-cysteine, sodiumhydroxide and propionic acid) (Beerens, 1990). Lactobacilli andenterobacteria were quantified in MRS agar (Merck, Frankfurt,Germany) and McConkey agar (Difco™), respectively. Incubationwas performed at 37 �C under anaerobic conditions using the anaer-obic jar with Anaerogen� for bifidobacteria or CO2 Gen� sachet forlactobacilli and enterobacteria. Number of cells was recorded ascfu/g of cecal material after 24 h incubation for lactobacilli or 72 hfor enterobacteria and bifidobacteria.

2.3.2. Histological analysis of caecum tissuesCaecum tissues were fixed in 10 % formalin. Tissue fragments

were imbibed in paraffin and stained with haematoxylin and eosin(H&E) for histological examination.

2.3.3. Short-chain fatty acid (SCFAs) analysisPropionic, butyric and acetic acids (SCFAs) were quantified in

cecal material as previously reported (Tzortzis, Goulas, Gee, & Gib-son, 2005). Briefly, cecal samples were mixed with MilliQ water ina proportion of 1:1.5 (v:v), centrifuged at 12000g for 10 min.Supernatants were then filtered through a 0.22 lm Millipore filter,type GV (Millipore, Bedford, MA) and analyzed by HPLC, using aprepacked Aminex HPX-87 H strong cation-exchange resin column(300 � 7.8 mm i.d.), fitted with an ion exclusion microguard refillcartridge (Bio-Rad Laboratories, Richmond, Calif.) in a Waters2695 Separation Module (Waters, Milford, MA) equipped with anautoinjector, a 2996 photodiode array detector (PAD) and the Em-power software were used for HPLC analysis. A sample of 20 ll waseluted with 0.005 mol/L sulfuric acid at 0.6 ml/min and 50 �C.SCFAs were identified and quantified by comparing retention timesand UV–visible spectral data to known standards.

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Fig. 2. Principal component analysis (PCA) biplot. Score and loading plots are superimpmeasured (correlation loading plot) correspond to fructooligosaccharides (FOS), dry m(antioxidant capacity). The scores representing each accession (different labels used) indAC) that are important in the discrimination are characterized by large loadings. Thus,discrimination among the different accessions. The closer an accession is to a particular

2.4. Statistical analysis

Quantitative data are mean ± standard deviation (SD) values.Data were analyzed using SPSS for Windows 14.0 (SPSS, Chicago,IL, USA). One-way analysis of variance (ANOVA) followed by pair-wise comparisons post hoc Duncan test (p < 0.05) were performed.For multivariate statistical analysis, principal component analysis(PCA) was performed on mean-centered and standardized datausing Unscrambler 9.8 (CAMO A/S, Trondheim, Norway).

3. Results and discussion

3.1. Antioxidant capacity (AC) and total phenolic compounds (TPC)

AC values for the 35 yacon accessions ranged from 23 to136 lmol TE/g DM or 3.2–20.1 lmol TE/g fresh weigh (FW) (Ta-ble 2). The highest values were found in ARB 5563, Y. BLANCOand DPA 07011 accessions, while the lowest values were foundin ARB 5125, AKW 5075 and ARB 5382. Previous studies have re-ported AC values of 1.25 lmol TE/g FW for yacon roots quantifiedby the DPPH assay (Mikami, Yamaguchi, Shinmoto, & Tsushida,2009). In general, the AC values found in the 35 yacon accessionswere within the range reported for other Andean tuber crops, suchas potato (Solanum sp.), mashua (Tropaeolum tuberosum), oca (Ox-alis tuberosa) and olluco (Ullucus tuberosus) (3.4–15.1, 3.8–39.2,6.5–19.8 and 1.9–6.1 lmol TE/g FW, respectively) (Campos et al.,2006), and similar to chicuru (Stangea rhizantha) (3.9 lmol TE/gFW), another FOS rich Andean crop (Campos et al., 2009).

TPC values varied from 7.9 to 30.8 mg CAE/g DM or 0.9–3.3 mg ofCAE/g FW. The highest values were found in DPA 07011, Y. BLANCO,and DPA 7001; while the lowest values were found in AJC 5189, ARB5564 and ARV 5073 (Table 2). These findings are consistent withprevious studies showing that TPC for yacon roots are around38 mg CAE/g DM (Yan et al., 1999), and 5.7–3.5 mg of gallicacid equivalent/g DM (Simonovska, Vovk, Andrenšek, Valentová &

FOS

DM

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15189 ARB 5184 DPA 07008 AMM 51635382 DPA 07007 ARB 5125 AKW 50757006 DPA 7002 ARB 5563 P 1385RA ARB 5537 P 1185 GOM 130 5136 DPA 07011 DPA 7003 AMM 5135

osed. Samples (score plot) correspond to the different yacon accessions. Variablesatter (DM), RS (reducing sugars), S (sucrose), TPC (total phenolic content) and ACicate differences for the 35 yacon accessions. The variables (FOS, DM, RS, S, TPC andthe further the variable from the origin, the more influential is that variable in thevariable is indicative of high positive correlation to that variable.

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Ulrichová, 2003). Likewise, values reported for other Andean cropswere within the range 0.47–3.31 mg CAE/g FW (Campos et al.,2006). Anthocyanins were not detected in the yacon accessions thatpresented either purple peel and/or purple spots in the flesh byusing a spectrophotometric method (Giusti & Wrolstad, 2001),most likely due to the low concentrations. Similarly, the yellow ororange flesh accessions contained very low and non detectableamounts of carotenoids, quantified as previously described (Talcott& Howard, 1999). This finding was related to the low lipophilic AC(0.05–0.35 lmol TE/g FM) content quantified by the ABTS assay(Arnao et al., 2001). In general, a high correlation between AC andTPC (r = 0.89, p < 0.01) was found, being indicative that phenoliccompounds are mainly responsible for the AC of yacon roots.

HPLC-PAD analysis performed for 5 yacon accessions showedsimilar phenolic profiles. Chromatograms at 320 nm showed 14peaks of hydroxycinamic acid derivatives, being one of them chlor-ogenic acid (5-O-caffeoylquinic acid) (Fig. 1). The chlorogenic acidcontent for the five accessions varied from 1.8 to 7.5 mg/100 g FW(15–24% of total phenolics quantified by HPLC-PDA). This wasconsistent with previous investigations that reported chlorogenic

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Fig. 3. Effects of yacon flour diet on (a) body weight, (b) body weight gain, and (c) fedifferent letters at each time point indicate statistically significant differences (p < 0.05)

acid (4.9 ± 1.3 mg/100 g FW) (Yan et al., 1999), and caffeic acidderivatives, mainly esters of caffeic acid with the hydroxy groupsof aldaric acid (Takenaka et al., 2003) as main phenolics identifiedin yacon roots. In addition, tryptophan was detected within therange 0.5–2.8 mg/100 g. These values were consistent with previ-ously reported values for yacon roots (1.46 ± 0.07 mg/100 g FW)(Yan et al., 1999).

3.2. Dry mater (DM), Reducing sugars (RS), sucrose (S), andfructooligosacharides (FOS)

The content of RS, S, and FOS based on DM are presented in Ta-ble 2 Results showed that DM varied from 7.5 to 19.1 g/100 g;accordingly RS varied from 19.7 to 75.9 g/100 g DM (3.5 to 8.5 g/100 g FW), S varied from 2 to 16.8 g/100 g DM (0.3 to 1.8 g/100 gFW), and FOS varied from 6.4 to 65.0 g/100 g DM (0.7 to 12.3 g/100 g FW). The highest FOS contents were found in AJC 5189 fol-lowed by AKW 5075 and AMM 5163 with values of 65.0, 51.3and 49.7 g FOS/100 g DM respectively (Table 2). The content ofRS in yacon accessions was inversely correlated to the FOS content

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ed efficiency (weight gain/food consumption). Data are mean values (n = 16 ± SD),.

Fig. 4. Photographs from histological sections of caecum from guinea pigs fed with experimental diets. (a) Increased cell density as demonstrated by the higher number ofcells with blue stained nucleus in the yacon flour diet (I), compared to the inulin (II) or the control diets (III). (b) Cross sections of caecum tissue showing the relatively highernumber of crypts in yacon flour diet (I) compared to inulin (II) or control (III) diets. Sections stained with H&E were observed with light microscopy using a Carl Zeissmicroscope (100� magnification).

D. Campos et al. / Food Chemistry 135 (2012) 1592–1599 1597

(r = �0.81, p < 0.01). Likewise, accessions with high content of FOSpresented high DM contents (r = 0.75, p < 0.01).

RS and sucrose were within the 22.3–88.7 g/100 g DM range;these values were consistent with previously reported values for10 greenhouse cultivated yacon cultivars collected from Peru,Ecuador, Argentina and Bolivia (Hermann et al., 1997).

In general, our results showed higher variability on RS, sucroseand FOS contents compared to previous studies (Lachman, Havr-land, Fernández, & Dudjak, 2004; Ohyama et al., 1990; Valentováet al., 2006). This finding might be attributed to the larger numberof accessions analyzed in this study. Variability in FOS content hasbeen related to the activity of enzymes involved in synthesis andhydrolysis of FOS such as sucrose: sucrose 1-fructosyl transferase(1-SST), fructan: fructan 1-fructosyl transferase (1-FFT) and fructan1-exohydrolase (1-FEH). Synthesizing enzyme activities (1-SST and1-FFT) were always higher in rhizophores than in the tuberousroots, while hydrolysing activity (1-FEH) predominated in the lat-ter (Itaya, de Carvalho, & Figueiredo-Ribeiro, 2002).

A good visualization multivariate tool is principal componentanalysis (PCA). The 35 accessions are displayed on a biplot (Fig. 2)and correlations can be directly assessed for the different variablesevaluated (RS, S, FOS, DM, TPC, AC). This biplot showed that yaconaccessions with high FOS content were mostly high in DM andlow in RS and S. Likewise, accessions with high TPC were also highin AC. Similarly, there seems to be a trend, yacon accessions withvery high FOS content displayed low AC values. This analysis is

useful to identify accessions with potential as source of bioactivecompounds that can help to prevent chronic diseases involving oxi-dative stress and glucose metabolism disorders.

3.3. Evaluation of in vivo prebiotic effect of yacon FOS

3.3.1. Effects of yacon flour on body weight and feed efficiencyIn general, guinea pigs equally accepted the diets rich in yacon

flour, inulin and control. No diarrhea symptoms and/or any otherhealth problems were observed during this experiment. Weightgain and average body weight are presented in Fig. 3(a and b). Ingeneral body weight was similar among the three experimentalgroups; however the yacon flour diet favoured weight gain at week8th. These results are consistent with previous studies reportingthat yacon flour diet did not inhibit the growth and weight gainof male Wistar rats (Lobo et al., 2007) or female Wistar rats fedwith FOS or galactooligosaccharides (GOS) (Anthony, Merriman,& Heimbach, 2006) when compared to control groups. Results offeed efficiency (ratio of weight gain to food consumption, Fig. 3c)showed that the yacon flour group was higher (p < 0.05) than theinulin group at the third and fourth week, while at the eighth weekit was higher than the control group (p < 0.05).

3.3.2. Effects of yacon flour on caecum histologyThe histological analysis of caecum tissue showed that yacon

flour promoted caecum cell growth (Fig. 4a), and increased depth

Table 3Microbiota (log10 CFU/g sample) and short chain fatty acids (SCFAs) (lmol/g) in cecalmaterial from guinea pigs fed with different experimental diets.

Yacon flour Inulin Control

MicrobiotaBifidobacteria 8.7 ± 0.26a 8.2 ± 0.68a 5.8 ± 0.73b

Lactobacilli 5.9 ± 0.05a 6.1 ± 0.39a 4.3 ± 0.41b

Enterobacteria 2.5 ± 0.06a 3.0 ± 0.53a 3.2 ± 0.84a

SCFAsAcetate 77.67 ± 13.98a 62.33 ± 9.52ab 51.41 ± 8.94b

Propionate 19.42 ± 2.45a 18.33 ± 4.38a 13.75 ± 3.06b

Butyrate 9.75 ± 2.91a 8.33 ± 1.63a 0.7 ± 0.11b

Total SCFAs 110.83 ± 40.17a 87.08 ± 20.27ab 62.08 ± 17.49b

Data are mean (n = 16) ± SD. Different letters within each microbiota group or SCFAgroup indicate significant differences (p < 0.05).

1598 D. Campos et al. / Food Chemistry 135 (2012) 1592–1599

and number of bifurcated crypts (Fig. 4b). These findings are con-sistent with a study that showed that a diet supplemented with5 or 7.5% FOS from yacon flour enhanced the enlargement of theabsorbing surface in the large intestine and the caecum wall withincreased number of bifurcating crypts in male Wistar rats (Loboet al., 2007). The stimulatory effects of yacon FOS on enlargementof caecum and caecum wall crypts might contribute to mineralabsorption with implications in bone density and maintenance ofhealthy bones (Lobo et al., 2007).

3.3.3. Effects of yacon flour on bacterial population and short-chainfatty acids (SCFAs) in cecal material

Results from bacteriological analysis of cecal material are pre-sented in Table 3. Concentrations (log10 CFU/g wet sample) of bif-idobacteria and lactobacilli were significantly higher (p < 0.05) insamples obtained from animals fed with yacon flour and inulincompared to the control group, while no significant differenceswere observed for enterobacteria for the three experimentalgroups (p > 0.05). These results are consistent with previous stud-ies showing that FOS from yacon are efficiently metabolized by bif-idobacteria and lactobacilli in vitro (Pedreschi et al., 2003) andin vivo using a mice model (Bibas Bonet et al., 2010). Both, inulinand yacon FOS are fructooligosacharides with different degree ofpolymerization (DP). In general, yacon FOS (DP = 2–10) (Pedreschiet al., 2003), and inulin (DP = 2–60) promoted the growth of bifido-bacteria and lactobacilli and stimulated the intestinal immune sys-tem with T cell activation and induction of IL-10 and IFN (BibasBonet et al., 2010).

During the last decades, much emphasis has been given to therole of dietary fiber, prebiotics and production of SCFAs in gastro-intestinal functions (Mortensen & Clausen, 1996; Wong, de Souza,Kendall, Emam, & Jenkins, 2006), and the onset of gastrointestinaldisorders, cancer, and cardiovascular diseases (Wong et al., 2006).Dietary fibers including FOS are substrates for the gut microflora.End products of the fermentation of these substrates are SCFAs(primarily acetate, propionate, and butyrate). These end productshelp to maintain the colonic mucosa by providing around 70% oftheir metabolic requirements (Cummings, Pomare, Branch, Naylor,& Macfarlane, 1987).

Higher values of SCFAs in cecal samples (Table 3) were observedfor the experimental groups fed with yacon flour and inulin dietscompared to the control (p < 0.05) and it was correlated with ahigher number of bifidobacteria and lactobacilli. Yacon flour dietincreased the production of acetate, propionate, butyrate and totalSCFAs by 51%, 41%, 1293%, and 78.5% compared to the control diet;while lower increases were observed for the experimental groupfed with the inulin diet (21%, 33%, 1090%, and 40%, respectively).These results are supported by a previous study with Sprague–Dawley rats demonstrating that a FOS-containing diet resulted in

higher cecal butyrate concentrations and lower pH compared withthe control diet, and this was accompanied by higher cecal bifido-bacteria and total anaerobes (Campbell, Fahey, & Wolf, 1997). An-other study reported that differences in SCFAs profiles varyaccording to the FOS degree of polymerization (short-chain ormedium-chain) and changes in gut microbiota (Sung, Choi, Cho,& Yun, 2006).

4. Conclusions

This study demonstrated the great variability in contents of bio-active compounds and antioxidant activity of 35 yacon accessions.Overall, among the 35 accessions of yacon, the FOS content was in-versely correlated with reducing sugars (RS), total phenolic content(TPC) and AC and positively correlated with dry matter (DM). Theaccessions identified for their enhanced content of FOS (AJC 5189,AKW 5075 and AMM 5163) might be of interest for the FOS nutra-ceutical industry. Only a few accessions with reasonable amountsof FOS and TPC were found (AMM 5135, SAL 136 and ARB 5027).These accessions might be of interest as accessions of enhancedcontent of novel health promoting compounds.

The in vivo study demonstrated that a yacon flour diet enhancedcaecum cell density and crypts, confirming the role of SCFAs assource of energy for colonic mucosa. The prebiotic effects of yaconflour were demonstrated by the FOS-stimulating effect on bifido-bacteria and lactobacilli growth and increased concentrations ofSCFAs. Overall, these results strongly suggest that yacon FOS con-sumption might play an important role in colonic healthmaintenance.

Acknowledgements

The authors thank Marco Ibarra-Castro, Ana Carrasco and Adel-ayda Pardo for their technical assistance. Dr. Carlos Arbizu from theInternational Potato Center (CIP), Lima - Perú, is kindly acknowl-edged for provision of the 35 yacon accessions. This work wassupported by the Consejo Nacional de Ciencia y Tecnología(CONCYTEC), Perú.

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